The invention concerns a process for the production of a polypeptide with suitable glycosylation by culturing eukaryotic cells and isolating the polypeptide from the culture medium or/and the cells. In this process the desired glycosylated polypeptide can be produced recombinantly with the aid of endogenous gene activation or be produced naturally by the cells.
The production of glycoproteins by culturing eukaryotic cells is generally carried out in commercial culture media. The addition of certain substrates to the culture medium may be necessary in order to achieve a desired glycosylation of the polypeptide. This is described in the Japanese laid-open patent document H6-292 592 for a batch process in small volumes ( less than 1000 ml), a low initial cell density (5xc3x97104 cells/ml) and a short culture period (48 h) with a human IgM antibody as an example. In this case another sugar such as fructose, mannose, galactose, N-acetylglucosamine, ribose, fucose, N-acetylgalactosamine etc. is used instead of the conventional glucose for the recombinant production of antibodies in mammalian cells e.g. CHO cells. In addition a multi-step culture process is disclosed in which the cells are firstly cultured in a medium containing glucose which is subsequently substituted by a medium containing another sugar.
However, the process described in the Japanese laid-open patent document H6-29592 has serious disadvantages. The concentration of the sugar in the culture medium changes continuously as a result of its consumption during the cell culture so that a constant high degree of glycosylation of the polypeptides is not guaranteed. Furthermore the batch process is unsuitable if a constant substrate concentration is necessary for a desired glycosylation since the initial concentrations of the substrates continuously decrease due to cell metabolism. Moreover the high sugar concentrations required for high cell densities and a constant high degree of glycosylation must already be provided at the start of the fermentation which would, however, inhibit the growth of the cells and hence limit the attainable cell density. Therefore an economical production of highly glycosylated polypeptides is not possible using the process described in the above-mentioned Japanese laid-open patent document.
The culture of eukaryotic cells by a batch process with feeding (fed-batch) in which nutrient solution is added during the culture is known. In this type of process it is possible to achieve a high cell density and a longer culture period by suitable feeding. An example is the continuous and limited feeding of the essential amino acid glutamine which leads to an improved cell growth (Ljunggren et al., Biotech. Lett. 12 (1990), 705-710). The aim of feeding glutamine is to reduce the formation of ammonium since ammonium is toxic for animal cells (Mirabet et al., Biotechnol. Bioeng. 56 (1997), 530-537).
Gawlitzek et al. (Biotechnol. Bioeng. 57 (1998), 518-528) describe that increased concentrations of ammonium ions or glucosamine in the medium of cultured eukaryotic BHK-21 cells lead to an increase of the complexity of N-linked carbohydrate structures in recombinant glycoproteins which are secreted by the cultured cells. However, this finding is inconsistent with the results of Borys et al. (Biotechnol. Bioeng. 43 (1994), 505-514) or Andersen and Goochee (Biotechnol. Bioeng. 47 (1995), 95-105) where an inhibition of glycosylation by elevated ammonium concentrations in the culture medium was found. Hence it becomes clear that the control of ammonium formation in the culture is only an isolated aspect and is therefore not sufficient for the economical production of proteins with suitable glycosylation.
The degree of glycosylation of polypeptides can greatly influence their biological activity. This is elucidated in the following erythropoietin (EPO) example. EPO is a human glycoprotein which stimulates the production of red blood cells. EPO only occurs in the blood plasma of healthy persons in very low concentrations so that it is not possible to provide larger amounts in this manner. EP-B-0 148 605 and EP-B-0 205 564 describe the production of recombinant human EPO in CHO cells. The EPO described in EP-B-0 148 605 has a higher molecular weight than urinary EPO and no O-glycosylation. The EPO described in EP-B-0 205 564 from CHO cells is now available in large amounts and in a pure form.
Furthermore the isolation of human EPO from the urine of patients with aplastic anaemia is known (Miyake et al., J. Biol. Chem. 252 (1977), 5558-5564).
Recombinant and urinary EPO are isolated as a mixture of various isoforms which are known to differ in their degree of sialylation. These EPO isoforms have different isoelectric points and can be separated by isoelectric focussing or capillary electrophoresis (see Tsao et al., Biotech. Bioeng. 40 (1992), 1190-1196; Nieto et al., Anal. Commun. 33 (1996), 425-427; Tran et al., J. Chromatogr. 542 (1991), 459-471; Bietot et al., J. Chromatogr. 759 (1997), 177-184; Watson et al., Anal. Biochem. 210 (1993), 389-393). The isoforms with the highest number of sialic acids have the highest specific activity, whereas those with the lowest number have the lowest activity (see e.g. Imai et al., Eur. J. Biochem. 194 (1990), 457-462; EP-A-0 428 267).
Takeuchi et al., (Proc. Natl. Acad. Sci. USA 86 (1989), 7819-7822) describe a relationship between the biological activity and the sialic acid content and the ratio of biantennary and tetraantennary carbohydrate structures. Takeuchi et al., additionally conclude that the N-acetyl-lactosamine disaccharide units present in the EPO carbohydrate structures do not correlate with the biological activity.
Fukuda et al., (Blood 73 (1989), 84-89) deal with the rate of elimination of EPO from the blood circulation which makes an important contribution to the biological activity and conclude that EPO with a relatively large number of N-acetyl-lactosamine units is more rapidly removed from the circulation than EPO without lactosamine units. Morimoto et al., (Glycoconjugate J. 13 (1996), 1093-1120) describe the separation of EPO isoforms by means of mono-Q chromatography so that the individual fractions are then only composed of a few isoforms. The investigations carried out on these fractions show an equidistribution of all structures in all fractions. No correlation was found between the content of biantennary or triantennary structures or the content of N-acetyl-lactosamine units and the specific activity.
Thus the said prior art shows that there is a general correlation of the biological activity with the sugar structure especially with regard to the content of sialic acids.
Surprisingly it was found that a continuous feeding according to requirements of carbohydrate-containing substrates during a high cell density fermentation or/and use of a mixture of at least 2 carbohydrates during culture enables a high yield of desired protein, such as EPO, with a high degree of glycosylation to be obtained.
Hence a first aspect of the invention concerns a process for isolating a glycosylated polypeptide from eukaryotic cells, wherein the eukaryotic cells are cultured in a suitable medium and the desired polypeptide is isolated from the cells or/and the culture supernatant wherein the process is characterized in that a mixture of at least 2 and preferably at least 3 carbohydrates is added to the culture medium.
The carbohydrates are preferably selected from monosaccharides and disaccharides such as glucose, glucosamine, ribose, fructose, galactose, mannose, sucrose, lactose, mannose-1-phosphate, mannose-1-sulfate and mannose-6-sulfate. Nutrient media are for example suitable which contain glucose or/and mannose or/and galactose. Particularly good results were obtained with nutrient media which contain a mixture of glucose, galactose and mannose for example in a mass ratio of 1:(0.5-3):(1-5) and in particular of 1:(0.7-2.4):(1.8-4.0) where each of the carbohydrates is particularly preferably used in the D(+) form. The total concentration of all sugars during the fermentation is preferably in a range of 0.1 to 10 g/l, particularly preferably in a range of 2 to 6 g/l in the culture medium. The carbohydrate mixture is preferably added dependent on the respective requirement of the cells as elucidated in more detail in the following.
A second aspect of the invention is a process for isolating a glycosylated polypeptide from eukaryotic cells in which the eukaryotic cells are cultured in a suitable medium and the desired polypeptide is isolated from the cells or/and the culture supernatant in which the process is characterized in that nutrients are added in a controlled manner and according to requirements during the culture which comprise at least one essential amino acid for the respective cultured cell line or/and at least one carbohydrate depending on the respective requirement of the cells.
In order to enable a demand-oriented addition of nutrients, the concentration of parameters which correlate with the nutrient requirement of the cells and their consumption rates are determined continuously or at suitable time intervals e.g. at least once daily. In this manner it is possible to quantitatively or/and qualitatively determine the nutrients required for the cell needs and to add them to the culture medium in an appropriate composition and amount. Such parameters can be nutrients or metabolic products of the cells such as the glutamine, the ammonium, the glucose or/and the lactate concentration, in particular the glutamine concentration.
As a result of the controlled and demand-oriented addition of nutrients it is possible to obtain a considerably improved glycosylation even with a high cell density fermentation (cell density at harvest greater than 10xc3x97105 cells/ml and preferably greater than 20xc3x97105 cells/ml) in large fermenters (volume greater than 1 l, e.g. 50 to 10,000 l).
The nutrients added according to this aspect of the invention comprise essential amino acids e.g. glutamine or/and tryptophan or/and carbohydrates and preferably in addition non-essential amino acids, vitamins, trace elements, salts or/and growth factors e.g. insulin. The nutrients preferably include at least one essential amino acid and at least one carbohydrate. These nutrients are preferably metered into the fermentation culture in a dissolved state. The nutrients preferably contain at least glutamine and carbohydrates especially a mixture of at least two carbohydrates as mentioned above. A mixture of glucose, galactose and mannose is particularly preferably used. In addition it is preferred that the nutrients are added according to needs over the entire growth phase of the cells i.e. dependent on the concentration of the selected parameters measured in the culture medium.
The quantity ratio of glutamine to carbohydrates in the nutrient solution is preferably selected such that it essentially corresponds to the consumption ratio in the fermenter. This enables a substantially constant concentration of the individual substrates to be maintained in the fermenter. The concentration of glutamine is preferably maintained at a value which is  less than 150 mg/l in the culture medium and prevents the development of an ammonium concentration of xe2x89xa72.3 mmol/l in the culture medium. During the fermentation the total concentration of the sugars is preferably in a range of 0.1 to 10 g/l, particularly preferably in a range of 2 to 6 g/l culture medium as already explained.
The nutrient solution that is used contains a mass ratio of glutamine to sugars which is preferably in a range of 1:3 to 20 and particularly preferably of 1:5 to 15 with reference to the total sugar. When a nutrient solution is used which contains glutamine as well as the three sugars glucose, galactose and mannose, the mass ratio of glutamine to the sugars is preferably 1:(1 to 3):(1 to 5):(2 to 8) and particularly preferably 1:(1.5 to 2.2):(1.5 to 3.6):(4 to 6).
The process according to the invention is fundamentally suitable for the production of any glycosylated polypeptides. However, polypeptides are suitable which carry one or several sialic acid residues since especially the degree of sialylation of the polypeptides can be increased by the process according to the invention. Furthermore it is also possible to influence the antennarity.
The process according to the invention is particularly suitable for the production of polypeptides that can be used therapeutically since their biological activity and hence also their pharmaceutical efficacy depends on the glycosylation and in particular on the degree of sialylation or/and on the antennarity. For example the glycosylated polypeptides can be selected from the group comprising physiologically active glycoproteins such as lymphokines, cytokines, immunoglobulins and hormones e.g. EPO, thrombopoietin (TPO), G-CSF, GM-CSF, interleukins, interferons, blood coagulation factors and tissue plasminogen activators. Polypeptides can be natural human polypeptides or recombinant muteins of such human polypeptides. The glycosylated polypeptide EPO is particularly preferred.
The cells used for the culture can in principle be any eukaryotic cells such as yeast cells or insect cells. However, the eukaryotic cells are preferably mammalian cells e.g. cells derived from the hamster such as CHO or BHK or in particular human cells. Furthermore it is preferred that the eukaryotic cells are continuous cell lines of animal or human origin such as the human cell lines HeLaS3 (Puck et al., J. Exp. Meth. 103 (1956), 273-284), Namalwa (Nadkarni et al., Cancer 23 (1969), 64-79), HT1080 (Rasheed et al., Cancer 33 (1973), 1027-1033) or cell lines derived therefrom. The desired polypeptide can be produced in the cultured cells
(a) by expression of a natural endogenous gene,
(b) by expression of an activated endogenous gene or/and
(c) by expression of an exogenous gene (recombinantly).
Cells are particularly preferred in which the desired polypeptide is produced by expression of an endogenous gene activated by homologous recombination e.g. the cell lines disclosed in the European Patent application EP 97 112 640.4 which are able to produce large amounts of EPO.
The cells can be cultured basically in any desired manner. However, culture as a suspension is preferred. Furthermore it is preferred that the cells are cultured in a medium containing a low serum content e.g. a maximum of 1% (v/v) or in particular in a serum-free medium e.g. in a serum-free, low-protein fermentation medium (cf. e.g. WO 96/35718). Examples of suitable culture media are basal media such as e.g. RPMI 1640, DMEM, F12 or eRDF containing appropriate additives. The process according to the invention allows a culture in large fermenters i.e. in a culture volume of more than 1 l, preferably more than 10 l, for example 50 l to 10,000 l. Furthermore the process according to the invention allows a high cell density fermentation which means that the concentration of the cells after the growth phase (i.e. at the time of harvest) is more than 10xc3x97105 cells/ml and particularly preferably more than 20xc3x97105 cells/ml.
The culture is preferably carried out as a repeated batch process with feeding according to requirements in which a portion of the culture broth is harvested after a growth phase and the remainder of the culture broth remains in the fermenter which is subsequently again filled up with fresh medium to the working volume. The process according to the invention enables the desired glycosylated polypeptide to be harvested in very high yields. Hence the concentration at the time of harvest is for example at least 30 mg and in particular at least 40 mg of the desired polypeptide per l culture medium.
Yet a further aspect of the invention is a process for isolating a glycosylated polypeptide from eukaryotic cells in which the eukaryotic cells are cultured in a suitable medium and the desired polypeptide is isolated from the cells or/and the culture supernatant, the process being characterized in that the culture is carried out at a temperature of xe2x89xa635.5xc2x0 C., preferably between 33 and 35.0xc2x0 C. It was surprisingly found that the proportion of polypeptides with the desired glycosylation can be considerably increased by lowering the temperature during the culture.